A large proportion of HIV related tuberculosis (TB) is caused by new Mycobacterium tuberculosis (Mtb) infection with rapid progression to active disease. Existence of host resistance to Mtb infection is supported by multiple lines of evidence, and understanding the mechanisms of resistance will be critical to decrease the burden of TB in HIV infected people. Mtb infection leads to latent TB infection (LTBI) which is inferred from T- cell immune assays. Since the absence of acquired anti-mycobacterial immunity defines resistance, innate mechanisms are critical to prevent Mtb infection and the establishment of LTBI. The hypotheses of our application are that alveolar macrophages (AMs), the first pulmonary cells that encounter inhaled Mtb bacilli, differ in their innate ability to resist establishment of Mtb infection, and tat inter-individual differences in innate AM resistance are controlled by host genetic factors. Hence, we propose to complement current methods of LTBI detection (TST, IGRA) with a cellular assay of infection resistance in AMs that will be used to optimize the resistance phenotype investigated in genetic studies. In aim 1 of our study, we propose to screen a large number of HIV-infected persons in South Africa (discovery sample) and Haiti (replication sample), and identify age-stratified groups of persons who are either TST/IGRA double positive or double negative. In aim 2, we will obtain AMs by broncho-alveolar lavage and derive monocyte-derived and induced pluripotent stem cells (iPSCs)-derived AM-like cells from blood of healthy and HIV-infected subjects. We will map Mtb-specific epigenetic modifications in the genome in presence and absence of HIV. In aim 3, we will derive induced pluripotent stem cells (iPSCs) from all enrolled South African subjects and use these iPSC AM-like cells for an assay that quantifies their innate resistance to Mtb infection by measuring Mtb uptake, intracellular growth, host cell apoptosis and production of cytokines as well as transcriptional and epigenetic changes. Next, we will conduct a quantitative trait locus (QTL) analysis and map the genetic variants that impact on gene expression levels and the quantitative read-outs of the functional AM assays. In aim 4, we will perform whole genome sequencing of the South African samples and engage in a search for rare variants with strong individual effects influencing a stringently defined infection resistance phenotype, employing both patient-centric and cohort-based designs. Genetic variants will be prioritized by a number of criteria, including their location in i) AM-specific epigenetic segments characteristic for HIV-infected subjects and either naive or infected AMs (aim 2), and (ii) regions known to be involved in infection resistance in HIV-negative subjects. Identified genetic resistance factors that replicate in the Haiti sample will be subjected to functional validation employing both subject PBMCs and subject iPSC AM-like cells. These experiments offer a powerful integrated approach to identify the molecular bases of Mtb infection resistance in HIV infected people, and point to a natural path forward through boosting of resistance factors in iPSCs cells by small molecule screening.